Given the failure of the existing approaches to control TB, it is imperative that improved strategies be developed for prevention. A panoply of M. tb. constituents that are targets of the humoral and cellular immune responses have been identified; approaches to determine which are potentially protective in humans are yet to be defined. To address this gap, this proposal is submitted by Drs. Ellner, Wallis, Boom, Stover, and Lathigra, investigators with complementary expertise in human in vitro model systems of the cellular immune response to mycobacteria; identification and characterization of mycobacterial targets of the human T-cell response; and cloning and expression of the relevant microbial genes and their introduction and expression in recombinant organisms. Working closely with leading researchers studying animal models of protective immunity, the goals of this consortium are three-fold: to develop a novel in vitro human model system that can predict which among characterized M. tb. antigens is/are likely to be potentially protective; to corroborate this prediction by demonstrating that rBCG organisms expressing the putative protective antigen show greater susceptibility to intracellular killing by the fully competent cellular immune response; and, ultimately, to seek confirmatory data in animal systems for mutual validation of the in vitro and in vivo models as predictive of protective immunity, and to confirm the status of the selected antigens as vaccine candidates. The Specific Aims are: 1. To develop an in vitro assay of T- cell dependent activation of monocytes (MN) to kill M. tb. as a model of protective immunity; to identify T-cell subpopulations and cytokines critical to this process; and to adapt the model to optimize screening of available and newly-defined antigens. Antigens of particular interest include the 30 kD alpha antigen (alpha-ag), the "P1" antigen, a target of the local immune response in tuberculous pleurisy, and the 58 kD MN- stimulatory protein; and, 2. To select antigens that are most stimulatory of T-cell dependent MN activation for cloning and expression as chimeric antigens in rBCG; then, to assess whether over-expression of the putative protective antigen(s) is associated with diminished capacity for intracellular survival in the presence of immune T-cells. Antigens defined in this in vitro model as likely to be protective will be studied both as vaccines (protein, subunit, and recombinant) for induction of protective immunity in guinea pigs and mice. Once validated, this approach will provide extensive data concerning the likely efficacy of currently defined mycobacterial products as vaccines and establish a paradigm for the study of future promising vaccine candidates.